Abstract The proposed research project continues and expands a line of investigation on the structure and function of thrombin and its zymogen precursors that has been continuously funded by the NIH since 1994. We now propose to expand our investigation of the molecular architecture of prothrombin with 19F NMR and cryo-EM, two powerful techniques never before used in the study of this important clotting factor. Our goal is to characterize the structural plasticity of prothrombin free and bound to its physiological activator prothrombinase and to significantly advance our molecular understanding of how this prototypic, multi-domain clotting factor functions as a substrate in the most important reaction of the blood coagulation cascade. Studies under specific aim 1 will characterize the conformational landscape of prothrombin and its derivatives prethrombin-2 and thrombin using 19F NMR. We hypothesize that the recently discovered open-closed equilibrium of prothrombin is controlled by the conformational properties of two critical residues of the protease domain, i.e., W547 in the active site and W468 in the flexible autolysis loop, and their cross-talk with the auxiliary Gla domain, kringles and sites of activation at R271 and R320. To test this hypothesis, we will carry out pioneering 19F NMR measurements targeting the nine Trp residues of the protease domain of prethrombin-2 and thrombin and will then extend this approach to full length prothrombin, free and bound to prothrombinase, by labeling the additional five Trp residues of the Gla domain and kringles. Site-specific labeling of W547, W468, R271 and R320 for 19F NMR measurements of environment and dynamics will be obtained by replacement with Cys and conjugation with BFTA. These measurements will report changes that accompany activation (prethrombin-2 vs thrombin), binding of prothrombinase (prothrombin free vs bound) and role of auxiliary domains (prothrombin vs prethrombin-2). Studies under specific aim 2 will solve the structures of prothrombin free and bound to prothrombinase by cryo- EM. We will test a mechanism of activation where prothrombin exists predominantly in the closed form when free and retains this conformation when bound to prothrombinase to promote cleavage at R320 and initiate the meizothrombin pathway. Meizothrombin then switches to the open form and promotes cleavage at R271 leading to the mature enzyme thrombin. We have developed reagents in quantities and purity to test this hypothesis by direct visualization with cryo-EM and obtained preliminary cryo-EM structures of prothrombin, cofactor Va and the prothrombin-factor Xai complex. Stable particles of the prothrombin-prothrombinase complex have been imaged by EM and are currently in the queue for cryo-EM data collection. Results from our proposed research project will significantly advance our basic knowledge of a key reaction of the coagulation cascade and will impact the study of other trypsin-like zymogens with modular assembly.